Mass Spectrometry

Research and Instrumentation

Contents

Ion Mobility Spectrometry-Mass Spectrometry

The MS Facility has four ion mobility enabled mass spectrometers.

Ion mobility spectrometry enables separation of analytes according to their shape and charge, allowing ions with the same molecular mass but different cross sectional area to be separated and detected.

Combining IMS with MS is a powerful technique used for investigating how the size and shape of biologically relevant molecules change under different stimuli or conditions.

IMS-MS can be used to measure cross sections of multimeric non covalent protein complexes for example during amyloidogenic fibril formation or the complex intermediates that form during virus capsid formation.

Conformational changes in the presence and absence of specific and non-specific ligands can be also be probed.

The instrumentation in the Facility uses Travelling Wave Ion Mobliity (TWIMS) where ions are propelled through a buffer-filled ion guide by a 'travelling wave' potential that is applied sequentially to ring electrodes.

As the ions traverse the ion guide propelled by the wave they undergo multiple collisions with the buffer gas. The shape and charge on the analytes will determine the magnitude of the interaction with the buffer gas, if this is sufficient it will retard the mobility of the analytes and cause it to 'roll over' the top of the wave.

In this manner, analytes are separated and subsequently detected by the mass spectrometer. With careful calibration of the TWIMS device the collisional cross sectional areas of analytes can be determined.

If IMS-MS could be applied to your research, please contact the Facility with some details about the project and we can arrange to discuss sample requirements and costing. Email mass-spec@leeds.ac.uk for more information.

Fast Photochemical Oxidation of Proteins (FPOP)

Photochemical labelling of proteins is one of the structural proteomics approaches being employed by the Facility to probe solution phase structure of proteins.

Mass spectrometry is used to map sites of covalent chemical modification to examine solvent accessibility and how this changes under different conditions.

This technique can be used to investigate conformational changes of a protein or protein complexes in the presence or absence of a small molecule for example or to determine the binding interfaces between individual proteins in a higher order complex.

Fast photochemical oxidation of proteins uses a solution of proteins in the presence of small amounts of hydrogen peroxide and radial scavenger glutamine.

When the solution is exposed to bursts of laser irradiation, the hydrogen peroxide breaks down to form hydroxyl radicals that quickly react with amino acid side chains on the proteins causing oxidation.

The speed of the oxidation reaction is much faster than any conformational change that may occur and the presence of the glutamine ensures a short lifetime of the hydroxyl radicals.

The position of the oxidations introduced to the molecules are then determined using a bottom-up proteomics approach.

Comparison of the levels of oxidation on the peptides under different conditions enables information about solvent accessibility to be inferred.

Hydrogen-Deuterium Exchange-MS (HDX-MS)

In the presence of deuterated water, hydrogen atoms with solvent accessibility can exchange with the deuterons.

Mass spectrometry can be used to detect the changes in mass corresponding to incorporation of deuterium.

This is a reversible process and care must be taken to avoid conditions that promote excessive back-exchange or scrambling of the position of the exchanged deuterons.

Using a refrigerated LC column module and online pepsin digestion, these detrimental process can be minimised.

Under these conditions exchange at the amide nitrogens on the protein backbone are probed. The major advantage of this technique over covalent modification is that different deuterium labelling times can be used which enables information on the kinetics of exchange, and thus information on the deuteron's environment, to be probed.

Cross Linking-Mass Spectrometry (XL-MS)

Bifunctional probes can be used to react specifically with certain amino acid side chain groups that are positioned in proximity to each other and within a distance defined by the length of the employed probe.

Reaction of the cross linking reagent with each side chain links the amino acids together chemically providing a snap shot of the conformation of the protein.

Using bottom up proteomics approaches, the cross linked peptides can be detected and charaterised providing a distance constraint between the two parts of the molecule which could potetially be far apart in the primary sequence.

Cross linking is complementary to other structural approaches such as NMR and X-ray crystallography. It is particulalry useful when sample amounts are limited or the system in question is intransigent to analyses by other techniques.